A sensitive assay for 06-alkylguanine-DNA alkyltransferase activity in cell or tumor extracts has been devised. The theoretical basis of the new assay lies in the observation that certain restriction enzymes will not cleave DNA containing methylated bases. Thus if a synethetic oligodeoxynucleotide with a restriction sequence containing 06-methylguanine were incubated with the restriction enzyme, this synthetic oligodeoxynucleotide shoud remain intact. However, if the guanine-06 methyl group were first removed by 06-alkylguanine-DNA alkyltransferase present in certain cell or tissue extracts the synthetic oligodeoxynucleotide would be cleaved by the restriction enzyme. The parental oligodeoxynucleotide and its restriction products are separated from each other and analyzed on denaturing polyacrylamide gels. The extent of cleavage by the restriction enzyme is a direct assay of the content of 06-alkylguanine-DNA alkyltransferase in the cell/tumor extracts. The assay has been tested against cell culture and xenograft tumor systems and has performed in a predictive manner, correctly predicting all the Mer- phenotype and 2 of 3 Mer+ phenotype. Furthermore the assay is quantitative and the number of molecules of the 06-alkylguanine-DNA akyltransferase per cell estimated using this assay agrees with those that have been published.